This invention relates to cyclic compounds that undergo nucleotide base pair-specific interactions with double stranded nucleic acids. The invention also concerns methods of using such cyclic compounds, as well as methods relating to their solid state synthesis.
None of the following discussion of the background of the invention, which is provided solely to aid the reader in understanding the invention, is admitted to be or to describe prior art to the invention.
The design of synthetic ligands capable of reading information stored in the DNA double helix has been a long-standing goal of chemistry and molecular biology. Cell-permeable small molecules, which target predetermined nucleotide sequences in double-stranded nucleic acids, particularly double-stranded DNA (dsDNA), are useful in regulating, or modulating, gene-expression. Oligodeoxynucleotides that recognize the major groove of double-helical DNA via triple-helix formation bind to a broad range of sequences with high affinity and specificity. See Moser, et al., Science, vol. 238:645-650 (1987), Duvalvalentin, et al., Proc. Nat""l Acad. Sci. USA, vol. 89:504-508 (1992), Maher, et al., Biochemistry, vol. 31:70-81 (1992). Although oligonucleotides and their analogs have been shown to interfere with gene expression, the triple helix approach so far has been limited to purine tracks and suffers from poor cellular uptake.
Another recent approach to targeting specific nucleotide sequences in dsDNA has involved molecules known as xe2x80x9cpolyamides.xe2x80x9d See U.S. Ser. No. 08/607,078, PCT/US97/03332, U.S. Ser. Nos. 08/837,524, 08/853,525, PCT/US97/12733, U.S. Ser. No. 08/853,522, PCT/US97/12722, PCT/US98/06997, PCT/US98/02444, PCT/US98/02684, PCT/US98/01006, PCT/US98/03829, and PCT/US98/0714. As described in the foregoing references, polyamides comprise polymers of amino acids covalently linked by amide bonds. Preferably, the amino acids used to form these polymers include N-methylpyrrole (Py) and N-methylimidazole (Im).
Wade, et al. (J. Am. Chem. Soc., vol. 114:8783-8794 (1992)) reported the design of polyamides that bind in the minor groove of dsDNA at 5xe2x80x2-(A,T)G(A,T)C(A,T)-3xe2x80x2 sequences by a dimeric, side-by-side motif; Mrksich, et al. (Proc. Natl. Acad. Sci. USA, vol. 89:7586-7590 (1992)), reported an antiparallel, side-by-side polyamide motif for sequence-specific recognition in the minor groove of dsDNA by the designed peptide 1-methylimidazole-2-carboxamide netropsin; and Trauger, et al. (Nature, vol. 382:559-561 (1996)) reported the recognition of a targeted dsDNA by a polyamide at subnanomolar concentrations. The particular order of amino acids in such polyamides, and their pairing in dimeric, antiparallel complexes formed by association of two polyamide polymers, determines the sequence of nucleotides in dsDNA with which the polymers preferably associate.
The development of pairing rules for minor groove binding polyamides derived from N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids provided a useful code to control target nucleotide base pair sequence specificity. Specifically, an Im/Py pair in adjacent polymers was fond to distinguish Gxe2x80xa2C from Cxe2x80xa2G and both of these from Axe2x80xa2T or Txe2x80xa2A base pairs. A Py/Py pair was found to specify Axe2x80xa2T from Gxe2x80xa2C but could not distinguish Axe2x80xa2T from Txe2x80xa2A. White, et al. (Biochemistry, vol. 35:12532-12537 (1996)) reported the effects of the Axe2x80xa2T/Txe2x80xa2A degeneracy of Py/Im polyamide recognition in the minor groove of dsDNA. White, et al. (Chem. and Biol. vol. 4:569-578 (1997)) reported the pairing rules for recognition in the minor groove of dsDNA by Py/Im polyamides and the 5xe2x80x2xe2x86x923xe2x80x2, Nxe2x86x92C orientation preference for polyamide binding in the minor groove of dsDNA.
More recently, it has been discovered that inclusion of a new aromatic amino acid, 3-hydroxy-N-methylpyrrole (Hp)(made by replacing a single hydrogen atom in Py with a hydroxy group), in a polyamide and paired opposite Py enables Axe2x80xa2T to be discriminated from Txe2x80xa2A by an order of magnitude. Utilizing Hp together with Py and Im in polyamides provides a code to distinguish all four Watson-Crick base pairs (i.e., Axe2x80xa2T, Txe2x80xa2A, Gxe2x80xa2C, and Cxe2x80xa2G) in the minor groove of dsDNA, as described in Table 1.
As discussed above, a number of different polyamide motifs have been reported in the literature, including xe2x80x9chairpins,xe2x80x9d xe2x80x9cH-pins,xe2x80x9d xe2x80x9coverlapped,xe2x80x9d xe2x80x9cslipped,xe2x80x9d and xe2x80x9cextendedxe2x80x9d polyamide motifs. Specifically, hairpin polyamides are those wherein the carboxy terminus of one amino acid polymer is linked via a linker molecule, typically aminobutyric acid or a derivative thereof to the amino terminus of the second polymer portion of the polyamide. Indeed, the linker amino acid xcex3-aminobutyric acid (xcex3), when used to connect first and second polyamide polymer portions, or polyamide subunits, Cxe2x86x92N in a xe2x80x9chairpin motif,xe2x80x9d enables construction of polyamides that bind to predetermined target sites in dsDNA with more than 100-fold enhanced affinity relative to unlinked polyamide subunits. See Trauger, et al., Nature, vol. 382:559-561 (1996), Swalley, et al., J. Am. Chem. Soc., vol. 119:6953-6961 (1997), Turner, et al., J. Am. Chem. Soc., vol. 119:7636-7644 (1997), Trauger, et al., Angew. Chemie. Int. Ed. Eng., vol. 37:1421-1423 (1997), Turner, et al., J. Am. Chem. Soc., vol. 120:6219-6226 (1998), Kelly, et al., Proc. Nat""l Acad. Sci. USA, vol. 93:6981-6985 (1996), Trauger, et al., J. Am. Chem. Soc., vol. 118:6160-6166 (1996), Geierstanger, et al., Nature Struct. Biol., vol. 3:321-324 (1996), Swalley, et al., Chem. Eur. J., vol. 3:1600-1607 (1997), and Trauger, et al., J. Am. Chem. Soc., vol. 120:3534-3535 (1998). Moreover, eight-ring hairpin polyamides (comprised of two four amino acid polymer portions linked Cxe2x86x92N) have been found to regulate transcription and permeate a variety of cell types in culture. See Gottesfield, J. M.; et al., Nature, vol. 387:202-205 (1997).
An H-pin polyamide motif, i.e., wherein two paired, antiparallel polyamide subunits are linked by a linker covalently attached to an internal polyamide pair, have also been reported. Another polyamide motif that can be formed between linked or unlinked polyamide subunits is an xe2x80x9cextendedxe2x80x9d motif, wherein one of the polyamide subunits comprises more amino acids than the other, and thus has a single-stranded region. See U.S. Ser. No. 08/607,078. In contrast, an xe2x80x9coverlappedxe2x80x9d polyamide is one wherein the antiparallel polyamide subunits completely overlap, whereas in a xe2x80x9cslippedxe2x80x9d binding motif, the two subunits overlap only partially, with the C-terminal portions not associating with the N-terminal regions of the other subunit. See U.S. Ser. No. 08/607,078.
The literature has also reported the synthesis of a six-ring Py/Im-containing cyclic polyamide (cyclo-(Im-Py-Py-xcex3-Py-Py-Py-xcex3)) that bound its designated five base pair (bp) dsDNA target sequence (5xe2x80x2-TGTTA-3xe2x80x2) at subnanomolar concentrations, and with 40-fold higher affinity relative to a hairpin analog (Im-Py-Py-xcex3-Py-Py-Py-Dp) containing the same amino acid pairings. See Cho, et al., Proc. Nat""l Acad. Sci. USA, vol. 92:10389-10392 (1995). It was postulated that closing the ends of the hairpin to form a cyclic compound would restrict conformational space for the DNA-binding molecule. Significantly, however, the hairpin analog more strongly bound to its match sequence versus single base pair mismatch sites by a factor of 20, whereas the cyclic polyamide bound match versus mis-matched sites with only 4-17-fold specificity, suggesting that an energetic price was paid by forming a cyclic molecule which had more restricted conformational flexibility as compared to non-cyclic polyamide motifs. Because of the discouraging thermodynamics of cyclic polyamides with regard to sequence specificity, cyclic polyamides appear to not have been investigated further, although the solid state synthesis of an 8-ring cyclic polyamide, cyclo-(Im-Py-Im-Py-xcex3-Im-Py-Im-Py-(G-Dp)-xcex3) was reported in PCT/97US/03332, as were the sequences of two apparently prophetic 8-ring cyclic polyamides. These sequences were cyclo(Im-Py-Im-Py-xcex3-Im-Py-Im-Py-(G-Dp)) and cyclo(Im-Py-Py-Py-xcex3-Im-Py-Py-Py-(G-Dp)). No biochemical characterization, e.g., target sequence binding affinity or sequence specificity, was provided for any of these three cyclic polyamides.
The present invention is based on the surprising and unexpected discovery of new and useful cyclic compounds, including cyclic polyamides, that interact in a nucleotide base pair-specific manner with desired target sequences in double-stranded nucleic acid molecules, particularly dsDNA, with affinities and specificities comparable to or better than naturally occurring DNA-binding proteins.
One aspect of the invention concerns cyclic compounds having molecular structures under physiological conditions that enable nucleotide base pair-specific interactions to be formed with high affinity and specificity with double-stranded nucleic acid molecules. Preferably, such molecules have an affinity for a specific sequence of nucleotide base pairs that is less than about 100 nM, preferably less than about 10 nM, and even more preferably, less than about 1 nM, as measured by DNase footprint titration See PCT/US97/03332. Similarly, such compounds have a sequence specificity, or prefer to bind to one sequence, the target sequence, by a factor of greater than about 2, preferably greater than about 5, and even more preferably greater than about 10, as compared to a sequence that differs by one nucleotide base pair.
Another aspect of the invention concerns cyclic compounds that contain at least seven units, at least one of which is a hydrogen bond donor or acceptor, configured in a cycle to form a molecular structure that interacts with specific nucleotide base pairs in a double-stranded nucleic acid molecule under physiological conditions. Specifically excluded from the cyclic compounds according to the invention are three cyclic polyamides having the following amino acid sequences: cyclo-(Im-Py-Im-Py-xcex3-Im-Py-Im-Py-(G-Dp)-xcex3), cyclo(Im-Py-Im-Py-xcex3-Im-Py-Im-Py-(G-Dp)), and cyclo(Im-Py-Py-Py-xcex3-Im-Py-Py-Py-(G-Dp)).
A xe2x80x9ccyclexe2x80x9d is a compound composed of covalently linked independent molecular units at least one of which is covalently linked directly (i.e., without a linker) or indirectly (i.e., through a linker) to at least two other independent molecular units of the cycle. A xe2x80x9ccovalent linkagexe2x80x9d refers to a chemical bond involving the sharing of one or more electrons between two or more atoms, and an xe2x80x9cindependent molecular unitxe2x80x9d or xe2x80x9cunitxe2x80x9d refers to a previously synthesized molecule that is incorporated into the cyclic compound during the latter compound""s synthesis. Preferred independent molecular units include, but are not limited to, molecules that are hydrogen bond donors or hydrogen bond acceptors, or molecules which become hydrogen bond donors or acceptors following incorporation into a cyclic compound according to the invention. Other preferred molecular units include amino acids, nucleosides, and carbohydrates.
A xe2x80x9chydrogen bond donorxe2x80x9d refers to a molecule or group of atoms including a hydrogen atom covalently linked to one electronegatively charged atom such that the hydrogen atom becomes electropositively charged and can thus be electrostatically attracted to interact with a second electronegative atom or group of atoms (in either case, the hydrogen bond acceptor). Representative hydrogen bond donors include a hydrogen covalently bonded to a nitrogen in an amide bond, and the 2-amino group of guanine. Representative hydrogen bond acceptors include the N3 of purines and Im, and the O2 of pyrimidines in dsDNA.
A xe2x80x9cnucleotide-specific interactionxe2x80x9d refers to a non-covalent interaction between a hydrogen bond donor or acceptor of a cyclic compound and a hydrogen bond acceptor or donor of nucleotide base in a double-stranded nucleic acid to form at least one hydrogen bond between a hydrogen bond donor or acceptor of a cyclic compound according to the invention with at least one of the nucleotides of a Watson-Crick base pair in a double-stranded nucleic acid. Preferably, at least one hydrogen bond is formed between each donor/donor or donor/acceptor pair of a cyclic compound and the adjacent nucleotide pair in the target sequence of the double-stranded nucleic acid, particularly dsDNA. A xe2x80x9cdonor/donor pairxe2x80x9d or a xe2x80x9cdonor/acceptor pairxe2x80x9d refers to a pair of hydrogen bond donors, or a hydrogen bond donor and acceptor, of a cyclic compound which reside on different polymer portions thereof but which are located proximate to one another in the cyclic compound such that each member of the pair interacts with a different member of the Watson-Crick nucleotide base pair with which one or both members of the donor/donor or donor/acceptor pair form one or more hydrogen bonds.
In preferred embodiments, a xe2x80x9ccyclic compoundxe2x80x9d refers to a compound that has two or more polymer portions or subunits, each of which polymer portions or subunits itself comprises multiple units at least one of which is a hydrogen bond donor or acceptor. In these embodiments, at a minimum the cyclic compound comprises two polymer portions. The first polymer portion comprises a polymer of at least three units, one or more of which acts as either a hydrogen bond donor or hydrogen bond acceptor. The second polymer portion comprises a polymer of at least four units, at least one of which also acts as either a hydrogen bond donor or hydrogen bond acceptor. The units which act as hydrogen bond or acceptors in either of the first or second polymers portions may be covalently linked to one another in a particular polymer, or they be separated by one or more intervening moieties, for example, a moiety which serves to restore the binding register to the cyclic compound. xe2x80x9cBinding registerxe2x80x9d refers to the alignment of hydrogen bond donors and acceptors of a polymer portion with their respective hydrogen bond acceptors and donors on one strand of a double-stranded nucleic acid. Preferred examples of such register restoring moieties include xcex2-alanine (xe2x80x9cxcex2xe2x80x9d), glycine, and other aliphatic chains which provide proper spacing between flanking hydrogen bond donors and/or acceptors of a polymer portion so as to enable hydrogen bond formation with corresponding hydrogen bond acceptors and donors of a strand of the double-stranded target nucleic acid.
To form a cyclic compound, the first and second polymer portions are attached at two or more locations, directly by covalent bonds between the polymers themselves, or indirectly, such as through the use of one or more linker molecules, or by a combination of direct and indirect attachment. As those in the art will appreciate, the resulting cyclic molecule may be symmetrical, in that each of the units (e.g., a hydrogen bond donors or acceptor) of one polymer portion align with a unit (e.g., a hydrogen bond donor or acceptor) of the other polymer portion. Alternatively, the cyclic compound may be asymmetrical, wherein at least one unit of one polymer portion does not align with a unit of the other polymer portion under physiological conditions in the presence of dsDNA.
In certain preferred embodiments of this aspect of the invention, at least one of the units, e.g., a hydrogen bond donor or acceptor, is an amino acid. An xe2x80x9caminoacidxe2x80x9d refers to any naturally occurring or synthetic molecule having an amino group, a carboxyl group, and an R group attached to the same or a different carbon atom. Representative examples of amino acids useful in the practice of this invention include the L- and D-forms of the twenty amino acids assembled into proteins in animals and plants, other aliphatic and aromatic amino acids, including pyrrole, hydroxypyrrole, and imidazole, and any chemical modifications or derivatives of any of the foregoing. Amino acids may serve as hydrogen bond donors (e.g., Py), hydrogen bond acceptors (e.g., Im), as register restoring moieties (also referred to herein as xe2x80x9cspringsxe2x80x9d), or as linkers. Particularly preferred for hydrogen bond formation are aromatic amino acids. An xe2x80x9caromatic amino acidxe2x80x9d is one wherein the R group comprises a ring having four or more members, preferably 5 or 6 members. In particularly preferred embodiments, the members of the ring members are independently selected from the group consisting of carbon, nitrogen, sulfur, and oxygen.
In other preferred embodiments, at least two of the hydrogen bond donors and/or acceptors of a polymer portion are covalently attached by an amide bond. Such cyclic compounds are referred to herein as cyclic polyamides. In certain particularly preferred embodiments, the first and second polymer portions comprise the same number of units, wherein a xe2x80x9cunitxe2x80x9d is a molecule that provides proper nucleotide-to-nucleotide spacing for a polymer of such units such that a hydrogen bond-stabilized non-covalent complex can be formed under physiological conditions between hydrogen bond donors and/or acceptors in the polymer and targeted hydrogen acceptors and/or donors in an adjacent nucleic acid strand of a double-stranded nucleic acid. Clearly, not all units within a polymer need to be hydrogen bond donors or acceptors, but each unit should provide spacing such that desired hydrogen bond formation can occur between specific hydrogen bond donors and/or acceptors in the polymer portion and corresponding hydrogen bond acceptors and/or donors in the nucleic acid strand with which the polymer forms a non-covalent complex. However, the polymer must contain a sufficient number of such units that are hydrogen bond donors or acceptors to enable hydrogen bond-stabilized non-covalent complex formation between a polymer of such units and a strand of a double-stranded nucleic acid.
The polymer portions, when linked to form cyclic compounds, may exhibit symmetrical motifs (e.g., an xe2x80x9coverlappedxe2x80x9d motif) or asymmetrical motifs (e.g., xe2x80x9cslippedxe2x80x9d or xe2x80x9cextendedxe2x80x9d motifs). In preferred embodiments, the polymer portions each comprise the same number of units, and may or may not contain the same number of hydrogen bond donors or acceptors. In particularly preferred embodiments, each polymer portion comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 units.
Certain preferred embodiments of this aspect of the invention concern polyamides, wherein each unit of the first and second polymer portions is an amino acid, preferably linked by amide bonds. Particularly preferred are those wherein each polymer portion comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, some, and preferably all, of which are selected from the group consisting of Py, Hp, Im, xcex2, and glycine. In some of these embodiments, the first and second polymer portions comprise equal numbers of amino acids, the particular order of which determines the nucleotide base pairs with which the cyclic compound will preferentially interact. When each polymer portion comprise an equal number of amino acids, a cyclic polyamide may have an xe2x80x9coverlappedxe2x80x9d or xe2x80x9cslippedxe2x80x9d binding motif. In other embodiments, each polymer portion comprises a different number of amino acids. Thus, the cyclic compound will typically have an xe2x80x9cextendedxe2x80x9d binding motif.
In many embodiments, the invention concerns cyclic compounds wherein each unit of the first polymer portion aligns with a unit of the second polymer portion to form a unit pair that preferably associates under physiological conditions with at least one, but not all, nucleotide base pairs selected from the group consisting of A/T, T/A, G/C, or C/G. A xe2x80x9cunit pairxe2x80x9d refers to two units, one from each of two different polymer portions, the align in a cyclic compound such that upon interaction with a double-stranded nucleic acid, each unit of the pair interacts (or if no interaction, aligns) with a different nucleotide in a Watson-Crick nucleotide base pair in a double-stranded nucleic acid. For example, if the unit pair interacts at A/T base pair, one unit of the unit pair interacts or otherwise aligns with A and the other unit aligns with T. Preferably, the unit pair preferably associates with at least one, but not all, of the four Watson-Crick base pairs (A/T, T/A, G/C, and C/G in dsDNA, and A/U, U/A, G/C, and C/G in RNA:DNA and RNA:RNA duplexes), thereby providing some level of sequence specificity. For example, it is known that Py/Py unit pairs prefer A/T or T/A base pairs in dsDNA, but not G/C or C/G base pairs. Particularly preferred are unit pairs that prefer one nucleotide base pair to the other three possible in a particular type double-stranded nucleic acid. For example, a Im/Py unit pair prefers G/C to the other three base pairs, whereas a Py/Im unit pair prefers C/G above the other three possibilities.
xe2x80x9cPhysiological conditionsxe2x80x9d refer to conditions found in vivo, or, alternatively, to in vitro reaction conditions intended to mimic or approximate those found in vivo. With regard to in vitro conditions which seek to approximate in vivo conditions, consideration should be given to pH, salt concentrations, buffering capacity, temperature, and such other parameters as may be deeded necessary in the particular circumstance. For purposes of illustration, physiological conditions in the context of interactions between acyclic compound according to the invention and dsDNA include those which are suitable for performing in vitro experiments which exploit the in vivo activity of an enzyme, for example, a DNA polymerase, an RNA polymerase, or an enzyme which modifies nucleic acids. Preferably, such enzymes will have at least 50%, more preferably, at least about 75%, and even more preferably, at least about 90%, of their optimal activity under the experimental conditions selected. As those in the art will appreciate, what constitutes physiological conditions in a given situation may depend on many factors, such as the type of organism being considered, the environment inhabited by the organism, etc.
In various embodiments of this aspect of the invention, a cyclic compound comprises at least one linker molecule. When used herein, xe2x80x9clinkerxe2x80x9d refers to a molecule used to covalently link two polymer portions of a cyclic compound. Preferably, two linkers are employed, although it may be desirable to include 1, 2, 3, or more additional linkers. Preferred linkers are those which comprise an aliphatic chain, for example, xcex2, glycine, and aminobutyric acid, with the latter being particularly preferred. Preferred cyclic compounds comprise two linkers comprising aliphatic chains, especially aminobutyric acid. Particularly preferred aminobutyric acids include xcex3-aminobutyric acid, particularly substituted derivatives thereof, for example, (R)-2,4-diaminobutyric acid. Linkers may be used to link terminal units of polymer portions to one another, or a terminal unit of one polymer to an internal unit of another polymer portion. Alternatively, linkers may be used to join two polymer portions by covalent linkages between internal units of each polymer portion.
Another aspect of the invention concerns a cyclic compound functionally associated (preferably by a covalent linkage) with an independent compound. Independent compounds can be any compound, with other cyclic compounds, proteins, nucleic acids, and polyamides (e.g., a polyamide molecule having a hairpin, H-pin, extended, overlapped, or slipped polyamide motif) being preferred.
The cyclic compounds of the invention interact with double-stranded nucleic acid with varying degrees of binding affinity and sequence specificity, depending on various factors, including compound size and composition, the targeted nucleotide base pair sequence, nucleic acid type, etc. Preferably, a cyclic compound will interact with its target nucleotide base pair sequence with an affinity, as measured by DNase footprint titration, of less than about 100 nM, preferably less than about 10 nM, more preferably less than about 1.0 nM, even more preferably less than about 0.1 nM. With regard to specificity (as measured in vitro under physiological conditions by comparing binding between a compound""s intended target sequence, i.e., a xe2x80x9cmatchxe2x80x9d site, and a test site equivalent to the target sequence except for a one nucleotide base pair difference, i.e., a xe2x80x9csingle base pair mismatchxe2x80x9d site), a cyclic compound of the invention should have at least about two-fold, preferably about 3-5 fold, more preferably 5-10-fold, and even more preferably greater than about 10-fold specificity for its match versus single base pair mismatch site.
When the double-stranded nucleic acid is dsDNA, cyclic compounds according to the invention may target specific molecular structures, for example, the major or minor groove, associated with a target nucleic acid sequence. Preferred cyclic compounds are those which interact with structures in the minor groove of dsDNA in the B-form. Cyclic polyamide compounds represent a particularly preferred class of such compounds.
Structurally, the cyclic compounds of the invention can be envisioned as comprising first and second polymer portions having the following formulas:
First polymer portion: 
wherein a xe2x89xa70 and b1, b2 and b3xe2x89xa71, x1, x2, x3, and x4 are units, one or more of which serve as hydrogen bond donors or acceptors, and one or more of which may be the same or different molecules, and z1, z2, and z3 each is a covalent linkage between adjacent units.
Second polymer portion: 
wherein c1, c2, c3 and c4xe2x89xa71, y1, y2, y3, and y4 each are units, one or more of which serve as hydrogen bond donors or acceptors, and one or more of which may be the same or different molecules, and zxe2x80x21, zxe2x80x22, and zxe2x80x23 each is a covalent linkage between adjacent units. In preferred embodiments, x2, x3, and x4 and y2, y3, and y4 form unit pairs that each preferably interact with at least one, but not all, Watson-Crick nucleotide base pairs, most preferably with only one such nucleotide base pair. The first and second polymer portions also typically comprise two termini. When the cyclic compound is a cyclic polyamide, one such terminus is an amino terminus, the other is a carboxy terminus. In particularly preferred embodiments, when contained in a cyclic polyamide, the polymer portions are arrayed in an anti-parallel manner.
Another aspect of the invention concerns pharmaceutical compositions comprising a cyclic compound according to the invention and a pharmaceutically acceptable carrier. Such compositions can be formulated into liquid or solid forms, and can be delivered by any appropriate route of administration. Preferred liquid formulations are aqueous solutions, suspension, slurries, gels, and emulsions. Preferred solid forms include pills, capsules, and powders (including lyophilized powders). Preferred routes of administration include parenteral (e.g., subcutaneous, intramuscular, intravenous, and interperitoneal) injection, transdermal delivery, inhalation, and oral delivery. Such compositions may also include additional components, for example, one or more other biologically active ingredients, liposomes (such as may be formed from various cationic lipids), etc. Such compositions may also be able to target delivery of a cyclic compound according to the invention to one or more particular cell or tissue types, or, with respect to eukaryotic cells, to an intracellular compartment containing double-stranded nucleic acid, e.g., a cell""s nucleus (such as by complexing the cycle with a nuclear localization signal).
Other aspects relate to methods of using the compounds according to the invention. One area of application relates to the modulation of gene expression. xe2x80x9cModulationxe2x80x9d refers to activating, increasing, enhancing, derepressing, reducing, decreasing, inhibiting, or preventing expression of a gene. Thus, some cyclic compounds positively affect, or up-regulate, gene expression, while others negatively affect, or down regulate, gene expression. A xe2x80x9cgenexe2x80x9d refers to genetic locus that encodes one or more gene products. As those in the art will appreciate, a gene can encode more than one gene product by virtue of differential mRNA splicing. Gene products include proteins (e.g., enzymes, receptors, antibodies, growth factors, and hormones) and RNA molecules, particularly tRNAs, ribosomal RNAs and other RNAs which are subunits of multi-component complexes (e.g., telomerase), and catalytic RNAs (e.g., ribozymes). To achieve the desired level of modulation in systems comprising cells, it is necessary to deliver a sufficient quantity of a cyclic compound according to the invention to the cells.
In some embodiments, the use of cyclic compounds according to the invention can modulate the expression of more than one gene. For example, more than one cyclic compound, each of which specifically modulates a particular gene, can be delivered. Alternatively, the cyclic compound may directly influence the expression of more than one gene. For example, if the cyclic compound targets a nucleotide base pair sequence found in a regulatory region of more than one gene, modulation of expression of multiple genes may occur. Alternatively, the cyclic compound may exhibit its expression-modulating effects indirectly, or by a combination of direct and indirect effects. For instance, if the cycle inhibits expression of a phosphatase that removes phosphates from a plurality of proteins, the expression of genes regulated by pathways that involve the phosphatase will be affected.
Certain embodiments of this aspect concern modulation of gene expression in vitro. xe2x80x9cIn vitroxe2x80x9d includes both in situ and cell-free environments (e.g., a cell extract or in a well-defined reaction medium). Thus, the compounds of the invention can be used to modulate gene expression in cultured cells, such as may be used in ex vivo therapy or research.
Other embodiments of this aspect relate to the modulation of gene expression in vivo, some of which concern therapeutic purposes. As used herein, a xe2x80x9ctherapeutic purposexe2x80x9d includes both therapy (i.e., treatment of an existing condition) and prophylaxis (i.e., prevention). Representative examples of a therapeutic purpose include treatment of a disease associated with aberrant expression of the gene of interest (as occurs in certain cancers and genetic diseases, for example), as well as treatment of a disease associated with the presence of a pathogen (e.g., a virus or a bacterial or eukaryotic pathogenic organism).
Cyclic compounds according to the invention can be used for therapeutic purposes in conjunction with a vast array of organisms, including both animals and plants. Preferred animals amenable to application of the therapeutic and prophylactic methods herein described include animals of agricultural importance, for example, avian (particularly poultry), bovine, equine, ovine, and porcine animals, companion animals such as dogs and cats, and humans. With regard to plants, preferred plants include those of agricultural importance, including cereals, grains, and grasses. Similarly, cyclic compounds according to the invention can be developed to control pests, e.g., certain insects and rodents.
Yet other aspects of the invention concerns methods for the solid phase synthesis of one or more cyclic polyamides. Such methods comprise providing a solid support (e.g., a polystyrene resin), protecting and activating the appropriate carboxamide monomers and/or dimers, sequentially adding the carboxamide monomers and/or dimers to the solid support, beginning with the carboxy terminal carboxamide residue, deprotecting the carboxamides following formation of the desired polyamide, releasing the polyamide from the solid support, and cyclizing the polyamide to form a cyclic polyamide. The resulting cyclic polyamide may then be purified, e.g., by reverse phase HPLC. Preferably, such purification results in a cyclic compound that has a purity of more than about 90%, preferably more than about 95%, particularly more than about 99%.